Hybrid SID, IM, and UVPD Methods for Complex-Down MS of Protein Complexes

为了完成它们的功能，许多蛋白质复合体必须首先与多个其他蛋白质形成动态复合体 蛋白质或结合伙伴。总体化学计量学、拓扑学以及亚基间和亚基内的特征 蛋白质和核蛋白复合体的接触及其组装/拆卸是至关重要的，因为这些 复合体调节关键的生物过程。天然质谱学(NMS)，特别是与 离子迁移率(IM)，以及碰撞诱导解离(CID)、紫外线等激活方法 光解离(UVPD)和表面诱导解离(SID)正在成为一种强大的技术 以此来研究这些复杂系统，并指导其他结构生物学工具的适当应用。 尽管NMS在结构生物学方面有希望，但商业仪器缺乏许多必要的工具来 充分描述这些复合体的特征。尚未商业化的SID已被证明是一种非常有用的 研究蛋白质复合体的工具，切割复合体中最弱的界面并产生亚 反映结构连通性的复合体。虽然IM在某些平台上可以商业化使用 在平台方面，分辨率往往不足以进行详细的结构研究。在tr&d1中，我们建议启用 将执行更高能量的SID，并在多台不同仪器上更有效地收集碎片离子 站台。在TR&D2中，我们建议在高分辨率Orbitrap仪器上开发高分辨率IM。在这 除了供应商原型IM之外，我们还建议将在TR&DS1和2中开发的技术结合在一起 设备，以便能够使用集成、高效的工作流程对蛋白质复合体进行全面表征。 SID和IM的耦合是必不可少的，因为当SID放在IM之前时，它允许构象信息 在完整的络合物和由SID产生的亚络合物上获得，即使当峰重叠在 M/z空间，从而能够构建结构模型。当SID放在IM之后时，它允许不同的构象 完整的复合体(如果存在)将被单独移动性地选择用于碎裂。除了联轴器侧 IM，我们建议将这些方法与UVPD相结合。这允许审问复杂的 与SID和IM的组装和亚基连接，以及共价碎裂(肽的测序 主干)来自UVPD。这种方法将有益于辨别配体结合位点以及 任何翻译后修改(PTM)。我们建议在多个仪器平台上实现这一点，包括 Waters Synapt G2(S)、Thermo(Q)Exactive和Bruker FTICR。使用多个平台是必要的 由于每个平台具有不同的质量分辨率、灵敏度和速度，因此某些平台会更适合 到特定的建筑群。因此，与多个平台整合后，可以定制实验以 感兴趣的综合体。使用多个平台也是至关重要的，因为它可以更好地向 更广泛的社区，他们可能只能访问这些平台中的一个。

In order to fulfill their functions many protein complexes must first form dynamic complexes with multiple other proteins or binding partners. Characterization of the overall stoichiometry, topology, and inter- and intra-subunit contacts of protein and nucleoprotein complexes, and their assembly/disassembly, is critical because these complexes regulate key biological processes. Native mass spectrometry (nMS), particularly in combination with ion mobility (IM), and activation methods such as collision-induced dissociation (CID), ultraviolet photodissociation (UVPD), and surface induced dissociation (SID), is emerging as a powerful technique with which to study these complex systems and for guiding appropriate application of other structural biology tools. Despite the promise of nMS for structural biology, commercial instruments lack many of the tools necessary to fully characterize these complexes. SID, which is not yet commercialized, has proven to be an incredibly useful tool in the study of protein complexes, cleaving the weakest interfaces in the complex and producing sub- complexes that are reflective of the structure’s connectivity. While IM is commercially available on some platforms, the resolution is often insufficient for detailed structural studies. In TR&D1, we propose to enable higher energy SID to be performed, with more efficient fragment ion collection on multiple different instrument platforms. In TR&D2 we propose to develop high-resolution IM on a high-resolution Orbitrap instrument. In this TR&D we propose to couple the technologies developed in TR&Ds1 and 2, in addition to vendor prototype IM devices, in order to enable the full characterization of protein complexes using integrated, efficient workflows. Coupling of SID and IM is essential because when SID is placed before IM it allows conformational information to be obtained on the intact complex and the subcomplexes produced from SID even when the peaks overlap in m/z space, enabling structural models to be built. When SID is placed after IM, it allows different conformations of the intact complex (if present) to be individually mobility-selected for fragmentation. In addition to coupling SID and IM, we propose to combine these approaches with UVPD. This allows for the interrogation of complex assembly and subunit connectivity with SID and IM, with covalent fragmentation (sequencing of the peptide backbone) from UVPD. This approach will be beneficial in discerning ligand binding sites along with the sites of any post-translational modifications (PTMs). We propose to do this on multiple instrumental platforms, including the Waters Synapt G2(S), Thermo (Q) Exactive, and Bruker FTICR. The use of multiple platforms is necessary as each platform has different mass resolution, sensitivity, and speed, and certain platforms will be better suited to certain complexes. Hence incorporation with multiple platforms allows the experiments to be customized to the complex of interest. The use of multiple platforms is also essential as it allows better dissemination to the wider community, who may have access to only one of these platforms.